Herbicides that inhibit 4-hydroxyphenylpyruvate dioxygenase (4-HPPD; EC 1.13.11.27), a key enzyme in the biosynthesis of the prenylquinones plastoquinone and tocopherols, have been used for selective weed control since the early 1990s. They block the conversion of 4-hydroxyphenylpyruvate to homogentisate in the biosynthetic pathway (Matringe et al., 2005, Pest Manag Sci., vol. 61:269-276; Mitchell et al., 2001, Pest Manag Sci. vol 57:120-128). Plastoquinone is thought to be a necessary cofactor of the enzyme phytoene desaturase in carotenoid biosynthesis (Boeger and Sandmann, 1998, Pestic Outlook, vol 9:29-35). Its inhibition results in the depletion of the plant plastoquinone and vitamin E pools, leading to bleaching symptoms. The loss of carotenoids, particularly in their function as protectors of the photosystems against photooxidation, leads to oxidative degradation of chlorophyll and photosynthetic membranes in growing shoot tissues. Consequently, chloroplast synthesis and function are disturbed (Boeger and Sandmann, 1998). The most important chemical classes of commercial 4-HPPD-inhibiting herbicides include pyrazolones, triketones and isoxazoles. The inhibitors mimic the binding of the substrate 4-hydroxyphenylpyruvate to an enzyme-bound ferrous ion in the active site by forming a stable ion-dipole charge transfer complex. Among 4-HPPD-inhibiting herbicides, the triketone sulcotrione was the first example of this herbicide group to be used in agriculture and identified in its mechanism of action (Schulz et al., 1993, FEBS Lett. Vol 318:162-166) The triketones have been reported to be derivatives of leptospermone, a herbicidal component from the bottlebrush plant, Callistemon spp (Lee et al. 1997, Weed Sci. Vol 45, 162-166).
Three main strategies are available for making plants tolerant to herbicides, i.e. (1) detoxifying the herbicide with an enzyme which transforms the herbicide, or its active metabolite, into non-toxic products, such as, for example, the enzymes for tolerance to bromoxynil or to Basta (EP242236, EP337899); (2) mutating the target enzyme into a functional enzyme which is less sensitive to the herbicide, or to its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP293356, Padgette S. R. et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant which are sufficient in relation to the herbicide, in view of the kinetic constants of this enzyme, so as to have enough of the functional enzyme available despite the presence of its inhibitor. The third strategy was described for successfully obtaining plants which were tolerant to HPPD inhibitors (WO96/38567). US2009/0172831 discloses nucleotide sequences encoding amino acid sequences having enzymatic activity such that the amino acid sequences are resistant to HPPD inhibitor herbicidal chemicals, in particular triketone inhibitor specific HPPD mutants.
To date, the prior art has not described HPPD-inhibiting herbicide tolerant plants containing microorganism-derived HPPD nucleic acid according to the present invention. What are also needed in the art are crop plants and crop plants having increased tolerance to herbicides such as HPPD-inhibiting herbicide and containing the microorganism-derived HPPD nucleic acid according to the present invention. Also needed are methods for controlling weed growth in the vicinity of such crop plants or crop plants. These compositions and methods would allow for the use of spray over techniques when applying herbicides to areas containing crop plant or crop plants
The present inventors have characterized various microorganism and plant species utilizing a mechanism of conferring resistance or tolerance to herbicides. Furthermore, the inventors have isolated and characterized the novel herbicide-resistance conferring HPPD from these microorganisms.
Row 1 shows non-transgenic control (wildtype plants)
Row 2 shows Arabidopsis plants comprising a polynucleotide encoding HPPD polypeptide of SEQ ID NO: 2 or 4 (Scenedesmus)
Row 3 shows Arabidopsis plants comprising a polynucleotide encoding HPPD polypeptide of SEQ ID NO: 30 (Acidobacterium)
Row 4 shows Arabidopsis plants comprising a polynucleotide encoding HPPD polypeptide of SEQ ID NO: 22 (Algoriphagus)
TABLE 1KEY TO SEQUENCE LISTINGNucleic acidAmino acidOrganismSEQ ID NO:SEQ ID NO:AccessionScenedesmus 1 2Scenedesmus short 3 4Helianthus WT 5 6Helianthus mutant 7 8Lemna 910Belliella1112I3Z863_BELBDNitritalea1314I5C6V4_9BACTPontibacter1516J1FG23_9BACTFerroplasma1718ZP_05569916Microscilla1920A1ZF21_9BACTAlgoriphagus2122A3HSH6_9BACTFrankia2324A8L2Y6_FRASNHerpetosiphon2526A9B1W0_HERA2Mucilaginibacter2728H1Y6Y7_9SPHIAcidobacterium2930C1F681_ACIC5Geodermatophilus3132D2SEF7_GEOOGScenedesmus codon-33optimizedScenedesmus short34codon-optimizedLemna codon-optimized35Scenedesmus codon-36E. coli expressionHelianthus codon37E.coli expressionHelianthus mutant codon38E.coli expressionHordeum vulgare3940Arabidopsis thaliana4142